The Exgen 500/DNA mixture was added to appropriate amounts of phe

The Exgen 500/DNA mixture was added to appropriate amounts of phenol red-free Opti-MEM (Invitrogen) and transferred to the wells. After transfection medium was removed and replaced by fresh DMEM medium (without DCC-FBS)

containing test substances or the solvent control. E2 10 nM and TCDD 1 nM served as the positive controls selleckchem for ERE- or XRE-mediated luciferase activity, respectively. After 20 h treatment cells were lysed with Reporter Lysis Buffer 1x (Promega). The microplate was then frozen at -80 °C for at least 30 min. Cells were scraped off, transferred into microtubes, and submitted to three sequential freezing-thawing cycles in liquid nitrogen and at 37 °C. Microtubes were centrifuged (5 min, 10 000 g, room temperature) and 10 μL of the lysate were pipetted into an opaque white 96-well plate. A volume of 50 μL luciferase assay reagent (Promega) was added to each well, the plate covered with an adhesive seal and immediately read in a microplate luminometer (TopCountNT, Packard). The β-galactosidase activity was determined using chlorophenol-red β-D-galactopyranoside (CPRG) (Roche), and the chlorophenol red product was measured on a microplate spectrophotometer at 570 nm (MRX Dynex). Protein levels were

measured on a spectrophotometer at 595 nm (MRX Dynex) according to the Bradford method [25]. Luciferase activity was normalized against β-galactosidase activity and protein contents and related to the respective positive controls. Total RNA was isolated with Selleckchem PI3K inhibitor the RNeasy Mini Kit (Qiagen). Samples were quantified spectrophotometrically via a NanoDrop 1000 Spectrophotometer (Thermo Fisher

Scientific). RNA (0.5 μg) was reverse-transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad) following DNase treatment (Desoxyribonuclease I, Amplification Grade, Invitrogen). Real-time PCR was performed in a total volume of 25 μL per reaction on an iCycler iQ Real-Time PCR Detection System with iCycler Software version 3.1 (Bio-Rad). Each PCR reaction contained 25 ng of the diluted cDNA, 12.5 μL of AbsoluteTM QPCR SYBR® Green Fluorescein Mix (Thermo Fisher Scientific), 200 nM of forward and reverse primers and pure water (qsp 25 μl). When a fluorogenic probe was used qPCR Master Mix no ROX (Eurogentec, Belgium) with a primer mix containing the primer pairs (300 nM/well) and the fluorogenic probe PTK6 (100 nM/well) were added instead. Primer sets were designed using the free software primer 3 (http://frodo.wi.mit.edu/) and purchased from MWG. Fluorogenic probes were designed and obtained from Eurogentec (primer sequences see Table 1). Optimized PCR consisted of 45 cycles at 95 °C for 15 seconds followed by amplification at 58 – 60 °C for 30 or 60 seconds. For real-time PCR using SYBR Green mix, a melting curve emerging in a gradient starting at the respective annealing temperature up to 90 °C in increasing steps of 0.5° C verified the single PCR product.